Siloxane column based liquid phase microextraction and process

ABSTRACT

A method for extracting an analyte in a sample is described. A sample and a solution in a microwave-extraction vial are microwave-heated and agitated. A vapor produced in the vial can be extracted into a liquid-phase medium contained in a porous membrane bag situated in the vial. The liquid-phase medium containing the vapor extract may then be analyzed for an analyte with gas chromatography-mass spectrometry.

STATEMENT OF ACKNOWLEDGEMENT

This project was prepared with financial support by the King Abdul AzizCity for Science and Technology through the Science and Technology Unitat King Fahd University of Petroleum and Minerals (project No.10-WAT1396-04), as part of the National Science Technology andInnovation Plan.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to a method of extracting an analyte froma sample using microwave-assisted headspace liquid-phasemicroextraction.

Description of the Related Art

The “background” description provided herein is for the purpose ofgenerally presenting the context of the disclosure. Work of thepresently named inventors, to the extent it is described in thisbackground section, as well as aspects of the description which may nototherwise qualify as prior art at the time of filing, are neitherexpressly or impliedly admitted as prior art against the presentinvention.

The Arabian Gulf area has undergone tremendous changes over the pastdecades in relation to management of water resources. According torecent estimations, daily production of desalinated water in gulfcountries reached up to 23 million cubic meters, and Saudi Arabiaproduces a lion's share of it, which is 11 million cubic meters[Roberts, D. A., el al., Impacts of Desalination Plant Discharges on theMarine Environment: A Critical Review of Published Studies. Water Res.2010, 44, 5117-5128—incorporated herein by reference in its entirety].The contaminants produced during the desalination have been extensivelyreported in the literature. Desalination contaminants have impacted themarine ecological environment, and as a result their detectableconcentrations were found in phytoplankton, invertebrates, and fish[Roberts, D. A., et al., Impacts of Desalination Plant Discharges on theMarine Environment: A Critical Review of Published Studies. Water Res.2010, 44, 5117-5128; Lattemann, S., et al., Environmental Impact andImpact Assessment of Seawater Desalination. Desalination 2008, 220,1-15—each incorporated herein by reference in its entirety]. Massivelosses of coral, plankton, and fish in the Hurghada region of the RedSea have also been attributed to desalination discharges [Hashim, A., etal., Impact of Desalination Plants Fluid Effluents on the Integrity ofSeawater, with the Arabian Gulf in Perspective. Desalination 2005, 182,373-393; Mabrook, B. Environmental Impact of Waste Brine Disposal ofDesalination Plants, Red Sea, Egypt. Desalination 1994, 97, 453-465—eachincorporated herein by reference in its entirety]. However, there existonly a few reports on the impact assessment and bioaccumulation ofdisinfection byproducts in biota samples. For example, the EasternProvince of Saudi Arabia, where the world's largest water desalinationplant is located, has not been evaluated to assess the impact ofdisinfection byproducts (DBPs) on biota. DBPs are unintentionallyproduced from the reactions of disinfectants with the natural organicmatter in the water [Richardson, S. D., et al., Water Analysis: EmergingContaminants and Current Issues. Anal. Chem. 2014, 86,2813-2848—incorporated herein by reference in its entirety]. Many of theDBPs have been shown to cause cancer, and reproductive and developmentaldisorders in laboratory animals [Drinking Water Treatment, U.S.Environmental Protection Agency, EPA Document No. 810-F-99-013,Cincinnati, Ohio, 1999—incorporated herein by reference in itsentirety]. They are also harmful to humans and are suspected carcinogenseven at low parts per billion (ppb) concentration levels.Trihalomethanes (THMs) and haloketones (HKs) are among the mostprevalent DBPs [Levesque, S., et al., Effects of Indoor Drinking WaterHandling on Trihalomethanes and Haloacetic Acids. Water Res. 2006, 40,2921-2930; Yang, X., et al., Factors Affecting Formation ofHaloacetonitriles, Haloketones, Chloropicrin and Cyanogen Halides duringChloramination. Water Res. 2007, 41, 1193-1200—each incorporated hereinby reference in its entirety]. For example, USEPA classifiedtrichloromethane (TCM), bromodichloromethane (BDCM), and tribromomethane(TBM) as carcinogens, while chlorodibromomethane (CDBM) was listed as apossible carcinogen [Xu, X., et al., Percutaneous Absorption ofTrihalomethanes, Haloacetic Acids, and Haloketones. Toxicol. Appl.Pharmacol. 2002, 184, 19-26—incorporated herein by reference in itsentirety]. Some toxicological effects of HKs are also reported. Moreprominently, chromosomal aberrations are associated withtrichloropropanone (TCP) [Blazak, W. F., et al., Activity of 1,1,1- and1,1,3-Trichloroacetones in a Chromosomal Aberration Assay in CHO Cellsand the Micronucleus and Spermhead Abnormality Assays in Mice. Mutat.Res. Toxicol. 1988, 206, 431-438—incorporated herein by reference in itsentirety], and 1,1-dichloropropanone (DCP) has been reported to reducecellular glutathione levels prior to cytotoxic effects [Merrick, B. A.,et al., Chemical Reactivity, Cytotoxicity, and Mutagenicity ofChloropropanones. Toxicol. Appl. Pharmacol. 1987, 91, 46-54—incorporatedherein by reference in its entirety]. Therefore, exposure to suchcompounds can lead to serious health implications.

The determination of various DBPs requires an efficient samplepreparation method prior to chromatographic analyses. During the lasttwo decades, different approaches of sample preparation have beenreported for DBPs. Liquid-liquid extraction and solid phase extractionare commonly used conventional approaches [US Environmental ProtectionAgency. 1995 a Method 551.1: EPA-600/R-95/131. USEPA Office of Researchand Development, National Exposure. Research Laboratory, Cincinnati,Ohio; US Environmental Protection Agency. 2003 Method 552.3: USEPAOffice of Ground Water and Drinking Water, Cincinnati, Ohio; Niri, V.H., et al., Fast Analysis of Volatile Organic Compounds and Disinfectionby-Products in Drinking Water Using Solid-Phase Microextraction-GasChromatography/time-of-Flight Mass Spectrometry. J. Chromatogr. A 2008,1201, 222-227—each incorporated herein by reference in its entirety].These methods have many shortcomings, including consumption of largevolumes of hazardous solvents, and furthermore, these time andlabor-extensive extractions lead to low recoveries. Therefore, it ishighly desirable to develop new extraction techniques for fast andaccurate quantitation of trace level concentrations of DBPs inbiological samples.

Microwave-assisted extraction (MAE) has a wide range of applications andovercomes many of the above-mentioned problems. It can be successfullyapplied to different biological samples such as plants and fish [Wang,S., et al., Design and Performance Evaluation of Ionic-Liquids-BasedMicrowave-Assisted Environmentally Friendly Extraction Technique forCamptothecin and 10-Hydroxycamptothecin from Samara of CamptothecaAcuminata. Ind. Eng. Chem. Res. 2011, 50, 13620-13627; Teo, C. C., etal., Development and Application of Microwave-Assisted ExtractionTechnique in Biological Sample Preparation for Small Molecule Analysis.Metabolomics 2013, 9, 1109-1128; Wang, H., et al., DynamicMicrowave-Assisted Extraction Coupled with Salting-out Liquid-LiquidExtraction for Determination of Steroid Hormones in Fish Tissues. J.Agric. Food Chem. 2012, 60, 10343-10351; Ma, Y., et al.,Microwave-Assisted Extraction Combined with Gel PermeationChromatography and Silica Gel Cleanup Followed by GasChromatography-Mass Spectrometry for the Determination ofOrganophosphorus Flame Retardants and Plasticizers in BiologicalSamples. Anal. Chim. Acta 2013, 786, 47-53; Dong, S., et al., FourDifferent Methods Comparison for Extraction of Astaxanthin from GreenAlga Haematococcus Pluvialis. Scientific World Journal. 2014, 2014, 1-7;Eskilsson, C. S., et al., Analytical-Scale Microwave-AssistedExtraction. J. Chromatogr. A 2000, 902, 227-250—each incorporated hereinby reference in its entirety]. Minimizing the degradation of volatileand semi-volatile compounds, shortening the extraction time, loweringhazardous solvent consumption, and simplifying the entire operation aremajor advantages of MAE [Eskilsson, C. S:, et al., Analytical-ScaleMicrowave-Assisted Extraction. J. Chromatogr. A 2000, 902, 227-250;Letellier, M., et al., Influence of Sediment Grain Size on theEfficiency of Focused Microwave Extraction of Polycyclic AromaticHydrocarbons. Analyst 1999, 124, 5-14; Mandal, V., et al., Design andPerformance Evaluation of a Microwave Based Low Carbon YieldingExtraction Technique for Naturally Occurring Bioactive Triterpenoid:Oleanolic Acid. Biochem. Eng. J. 2010, 50, 63-70; Ma, W., et al.,Application of Ionic Liquids Based Microwave-Assisted Extraction ofThree Alkaloids N-Nornuciferine, O-Nomuciferine, and Nuciferine fromLotus Leaf. Talanta 2010, 80, 1292-1297—each incorporated herein byreference in its entirety]. The combination of headspace extraction withMAE is an uncommon approach; however, to perform headspace extraction,instrumental modifications are required which makes it more tedious[Yeh, C.-H., el al., Headspace Solid-Phase Microextraction Analysis ofVolatile Components in Phalaenopsis Nobby's Pacific Sunset. Molecules2014, 19, 14080-14093—incorporated herein by reference in its entirety].

In view of the forgoing, one objective of the present disclosure is toprovide a single step microwave-assisted headspace liquid-phasemicroextraction (MA-HS-LPME) method for the isolation and subsequentdetermination of analytes such as THMs and HKs in biota samples. In oneaspect of this method, a PTFE ring is placed inside an extraction vialto support a solvent-containing porous membrane envelope or bag, andthus, no changes in the microwave instrument are required.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect, the present disclosure relates to a methodfor extracting an analyte in a sample.

The method involves microwave-heating and agitating the sample and asolution in a microwave extraction assembly, which comprises a microwaveextraction vial with a headspace portion and a bottom portion. Thebottom portion optionally contains a stir bar, and the headspace portioncontains a porous membrane bag. The porous membrane bag encapsulates aliquid-phase extraction medium. The sample and solution are located inthe bottom portion of the microwave extraction vial and do not contactthe porous membrane bag. The microwave-heating and agitating produces avapor in the headspace portion.

The method also involves extracting the analyte from the vapor toproduce a vapor extract within the liquid-phase extraction medium in themembrane bag.

In one embodiment of the method, the method also involves feeding thevapor extract to a gas chromatograph-mass spectrometer (GCMS) to detectand/or quantify the analyte.

In one embodiment of the method, the sample is derived from an organismthat lives in a body of water.

In one embodiment of the method, the weight of the sample relative tothe volume of the liquid-phase extraction medium is 0.04-100 g/mL.

In one embodiment of the method, the solution is an acid.

In one embodiment of the method, the solution is an acid, and themicrowave-heating and agitating simultaneously digests the sample andextracts the analyte.

In one embodiment of the method, the acid is at least one selected fromthe group consisting of nitric acid, hydrochloric acid, hydrofluoricacid, sulfuric acid, and perchloric acid.

In one embodiment of the method, the sample comprises the analyte and aninternal standard.

In one embodiment of the method, the porous membrane bag is positioned1-10 cm above the sample.

In one embodiment of the method, the weight of the sample relative tothe volume of the microwave extraction vial is 0.5-100 g/L.

In one embodiment of the method, the liquid-phase extraction medium isan organic solvent.

In one embodiment of the method, the liquid-phase extraction medium isan organic solvent which is at least one selected from the groupconsisting of benzene, cyclohexane, hexane, toluene, iso-octane,heptane, and decane.

In one embodiment of the method, the porous membrane bag comprises atleast one polymer selected from the group consisting of polypropylene,polyethylene, nylon, polyvinylidene fluoride, and polyethersulfone.

In one embodiment of the method, the porous membrane bag comprises aporous membrane having a 10-200 μm wall thickness.

In one embodiment of the method, the microwave extraction assemblyfurther comprises a ring within the headspace portion of the microwaveextraction vial to support the porous membrane bag.

In one embodiment of the method, the sample and solution aremicrowave-heated to a temperature of 40-100° C.

In one embodiment of the method, the sample and solution aremicrowave-heated and stirred for 1-25 min.

In one embodiment of the method, the analyte is a trihalomethane or ahaloketone.

In one embodiment of the method, the analyte is a trihalomethane or ahaloketone and the trihalomethane or haloketone, the analyte is detectedand/or quantified in the range of 0.02-200 ng per g of sample.

In a related embodiment, where the analyte is a trihalomethane or ahaloketone, the trihalomethane is bromodichloromethane, tribromomethane,trichloromethane, dibromochloromethane, or combinations thereof, and thehaloketone is 1,1-dichloro-2-propanone, 1,1,1-trichloro-2-propanone, orboth.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a microwave-heating source containing a microwave extractionassembly.

FIG. 2 is a microwave extraction assembly.

FIG. 3 is a graph showing the recoveries of the six disinfectionbyproducts (DBPs) of interest when using four different solvents as theliquid-phase extraction medium.

FIG. 4 is a graph showing the percent recoveries obtained for biologicalsamples spiked with 20 ng of each DBP per g sample when the porousmembrane bag has a different height (membrane depth) above the sampleand solution.

FIG. 5 is a graph showing the recoveries obtained for biological samplesspiked with 20 ng of each DBP per g sample when the extraction iscarried out at different temperatures.

FIG. 6 is a graph showing the recoveries obtained for biological samplesspiked with 20 ng of each DBP per g sample when the extraction iscarried out for different lengths of time.

FIG. 7 is a graph showing the recoveries obtained for biological samplesspiked with 20 ng of each DBP per g sample when different amounts ofsample are used.

FIG. 8 is a graph showing the DBPs recoveries obtained for each type ofbiological sample spiked with 20 ng of each DBP per g sample.

FIG. 9A is a GCMS chromatogram of DBPs extracted from fish scalesamples.

FIG. 9B is a GCMS chromatogram of DBPs extracted from fish scale samplesthat were spiked with 20 ng each DBP per g sample.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Embodiments of the present disclosure will now be described more fullyhereinafter with reference to the accompanying drawings, in which some,but not all embodiments of the disclosure are shown.

The present disclosure will be better understood with reference to thefollowing terms and meanings set forth below. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art.

According to a first aspect, the present disclosure relates to a methodfor extracting an analyte in a sample. The analyte may be an herbicide,a fungicide, a pesticide, a drug, a steroid, a microbial toxin, ametabolite, a disinfection byproduct, or some other small organicmolecule. In one embodiment, the analyte may be a disinfectionbyproduct. As used herein, the term “disinfection byproduct” is acompound produced from the reaction of a water disinfectant with anorganic impurity in an aqueous solution. Generally, the disinfectionbyproduct is a contaminant present in treated water or waste water thathas been processed in a desalination plant, a water treatment plant, ora pool. Organisms exposed to the contaminants may sequester thedisinfection byproducts in their tissues and organs. Disinfectionbyproducts may include iodoacetic acid, N-nitrosodimethylamine (NDMA),nitrogentrichloride, chloramine, halonitromethanes, haloacetonitriles,haloamides, halofuranones, nitrosamines, trihalomethanes, andhaloketones. A preferred embodiment of the method relates to detectingand/or quantifying trihalomethanes and haloketones, more specifically,the trihalomethanes bromodichloromethane (BDCM) (I), tribromomethane(TBM, or bromoform) (II), trichloromethane (TCM, or chloroform) (III),and dibromochloromethane (DBCM) (IV), and the haloketones1,1-dichloro-2-propanone (DCP) (V) and 1,1,1-trichloro-2-propanone (TCP)(VI).

Another embodiment of the method is concerned with detecting and/orquantifying an analyte in a sample derived from an organism that livesin a body of water. For example, the organism may be a fish, an alga, aseaweed, a turtle, a dolphin, an echinoderm, a squid, a ray, an insect,or a crustacean, and the body of water may be an ocean, a bay, a river,a lake, a swamp, or a pond. A sample derived from an organism may be apiece of external tissue, such as a piece of skin, scale, or leaf, or aninternal tissue such as a piece of muscle or liver, or the entireorganism. In an alternative embodiment, the sample may be water takenfrom a body of water such as those mentioned previously, or from theprocessed water or wastewater of a water treatment plant, sewagetreatment plant, or desalination plant. The sample may also be watertaken from a treated artificial body of water, such as a pool, fountain,bath, aquarium, or hot tub. The sample may also be water taken fromother natural environments such as groundwater and rainwater. The samplemay contain one analyte, or it may contain more than one. Where thesample contains more than one analyte, the molar ratio between any twomay range from 100:1 to 1:100, preferably 20:1 to 1:20, more preferably5:1 to 1:5. In a preferred embodiment, the analyte is a disinfectionbyproduct.

The method involves microwave-heating and agitating the sample and asolution in a microwave extraction assembly. FIG. 1 shows a microwaveextraction assembly 10 inside a microwave-heating source. FIG. 2 showsan embodiment of a microwave extraction assembly. The microwaveextraction assembly may comprise a microwave extraction vial 12 that mayhave a headspace portion 16 and a bottom portion 18. The bottom portionmay comprise a sample 26 and a solution 28. The vial may comprise glass,ceramic, or polytetrafluoroethylene (PTFE) and have an interior volumeof 20-300 mL, preferably 50-200 mL, more preferably 80-150 mL. The vialmay have a round bottom, a flat bottom, or a pointed bottom, and mayhave side walls that are curved, sloped to the bottom, or straight upand down. In one embodiment, the weight of the sample relative to thevolume of the microwave extraction vial may be 0.5-100 g/L, preferably2-80 g/L, more preferably 10-60 g/L. The vial may also have a closure 14in order to contain vapor and/or pressure produced by themicrowave-heating, or the vial may be open to atmospheric pressure buttopped by a vapor condenser. In a preferred embodiment, the vial is madeof PTFE and has a 100-125 mL volume with a closure.

The solution may include one or more of water, a salt solution, anorganic solvent, a cell lysis reagent, or an acid. The salt of the saltsolution may be sodium sulfate or sodium chloride. This organic solventmay have a dielectric constant of at least 5, preferably at least 10,more preferably at least 20 for efficient microwave-heating, and aboiling point of 50-100° C., preferably 60-90° C., more preferably65-80° C. The organic solvent may be an alcohol, a polar aproticsolvent, a non-polar solvent, or mixtures thereof. Examples of alcoholsinclude ethanol, methanol, and 2-propanol; examples of polar aproticsolvents include acetone, dimethyl sulfoxide (DMSO), acetonitrile,dimethylformamide (DMF), and ethyl acetate; examples of non-polarsolvents include hexane, pentane, benzene, 1,4-dioxane, and diethylether. The organic solvent may be added to water, such as mixing ethanoland water. The organic solvent may be added to water to a 20-80 vol %,preferably 30-70 vol %, more preferably 40-60 vol % concentrationrelative to the water and organic solvent mixture. A cell lysis reagentmay be present in the solution in order to disrupt the cell wall and/orcell membrane of a biological sample and release analyte. These reagentsmay be detergents such as sodium dodecyl sulfate and/or Triton X-100,salts such as sodium chloride, potassium chloride, and/or ammoniumsulfate, and/or a base such as sodium hydroxide and/or potassiumhydroxide. In one embodiment, the solution comprises an acid, forexample formic acid, benzoic acid, acetic acid, phosphoric acid,hydrobromic acid, hydroiodic acid, nitric acid, hydrochloric acid,hydrofluoric acid, sulfuric acid, and/or perchloric acid. Preferably theacid is nitric acid, hydrochloric acid, hydrofluoric acid, sulfuricacid, and/or perchloric acid; more preferably the acid is nitric acid.

In one embodiment, the sample may be mechanically processed prior tomicrowave-heating and agitating. This processing may lyse the cells of abiological sample, and may comprise sonication, extrusion, grinding,liquid homogenization, blending, scraping, slicing, centrifuging,drying, and/or freeze-thawing. In an alternative embodiment, cells of abiological sample may be ruptured by osmotic pressure.

In one embodiment, the concentration of the acid in the solution is 20mM-1 M, preferably 50mM-750 mM, more preferably 80-200 mM. A sufficientvolume of acid may be added to total 0.1-1 mmol, preferably 0.2-0.8mmol, more preferably 0.4-0.6 mmol acid per g sample.

In a preferred embodiment, the solution is 80-200 mM, or 90-120 mM, orabout 100 mM nitric acid, and a sufficient volume is added to result in0.45-0.55 mmol nitric acid per g of sample.

In an alternative embodiment where the sample may be processed water ora sample taken from a body of water, the solution may be omitted.

In one embodiment, the sample may comprise an internal standard. Theinternal standard may be present in the sample at a concentration of0.02-200 ng, preferably 0.5-100 ng, more preferably 1-70 ng per gsample. The internal standard may be 1,2-dibromopropane,2-benzoxalinone, 1-bromo-4-fluorobenzene, 1,1,1-trichloroethane,fluorobenzene, 4-bromofluorobenzene, trichloroethylene,1,4-dichlorobenzene, and/or 1,2-dichlorobenzene. The internal standardmay be a purified form of a previously mentioned disinfection byproductthat is known not to already exist in the sample. Or, the purifieddisinfection byproduct may be used as an internal standard in a samplethat contains the same compound by using standard additions of thepurified disinfection byproduct. Additionally, the internal standard maybe another compound that has no interaction with the sample, solution,or existing analytes, in terms of adsorbing to the sample or reactingwith other substances. Where an internal standard is added to a samplebefore undergoing extraction, the internal standard may show anextraction recovery of at least 80%, preferably at least 90%, morepreferably at least 96%. The internal standard and an analyte ofinterest may have boiling points that differ by less than 40° C.,preferably less than 30° C., more preferably less than 20° C. Theinternal standard may have similar partitioning properties compared withan analyte of interest. For example, where K_(ow) is the octanol-waterpartition coefficient of a compound, the values of log(K_(ow)) of aninternal standard and an analyte of interest may differ by less than2.0, preferably less than 1.5, more preferably less than 1.0.Preferably, though, the properties of the internal standard and analyteof interest are not so similar as to cause overlapping signals in thegas chromatogram or the mass spectra.

In one embodiment, the sample and solution are microwave-heated andstirred for 1-25 min, preferably 5-20 min, more preferably 10-15 min.The microwave power may be 10-1000 W, preferably 50-500 W, morepreferably 80-300 W. The microwave radiation may be delivered to thebottom portion of the vial, or to the entire microwave extractionassembly. The microwave radiation may be delivered continuously, orintermittently. In one embodiment, the microwave-heating may increasethe internal pressure of the microwave extraction vial. The internalpressure may be less than 40 bars, preferably less than 25 bars, morepreferably less than 10 bars. In one embodiment, the sample may beheated to and maintained at a temperature of 40-100° C., preferably60-90° C., more preferably 75-85° C. As shown in FIG. 1, amicrowave-heating source may be able to accommodate more than onemicrowave extraction assembly, and thus, more than one extraction may beperformed simultaneously. In an alternative embodiment, the sample maybe heated first, and then stirred without additional heating.

In one embodiment, the sample is heated by a means other thanmicrowave-heating, such as a hot water bath, a steam bath, a heat gun, aheating block, an oven, a wire heating element, an ultrasonicator, or byother non-microwave electromagnetic irradiation sources, such as aninfrared laser. Additionally, the sample may be heated simultaneously bytwo heat sources, such as microwave radiation and a hot water bath.Alternatively, the sample may be preheated, such as in a hot water bath,prior to microwave-heating and agitating. The sample may be heated witha variable power to maintain a constant temperature, or the sample maybe heated at a constant power with a variable temperature. Thetemperature of the sample may be inferred from a temperature probeplaced in the bottom portion or the headspace portion of the vial, or onan exterior surface of the vial. In the embodiment where a bath or ovenis used as a heating vessel, the temperature may be inferred from atemperature probe in the same heating vessel.

In one embodiment, the sample and solution are agitated by shaking,tilting, vortexing, or sonicating the vial, or by stirring the sampleand solution with a stir bar, a stirring rod, or an impeller.Preferably, the sample and solution are stirred with a stir bar 30located in the bottom portion of the vial. The stir bar may be stirredat a rate of 30-1000 rpm, preferably 60-700 rpm, more preferably 100-500rpm. The ideal stirring rate may be the highest speed that agitates thesample and solution without splashing the sample and/or solution intothe headspace portion, and this rate may depend on the type of sample,size of stir bar, and volumes of sample, solution, and microwaveextraction vial. In one embodiment, the sample and solution are heatedand stirred at rates that do not splash the sample and/or solution intothe headspace portion by the formation of heated vapor bubbles.

The sample may react with the solution during the microwave-heating andagitating step. In an embodiment where the solution is an acid and thesample is derived from an organism, the acid may digest the cells of thesample and release an analyte or analytes contained in the sample. Thisacid digestion may be accelerated by the microwave-heating and/oragitating. Alternatively, the acid may be added and allowed to reactwith the sample prior to microwave-heating and agitating. Themicrowave-heating and/or agitating may cause molecules of sample,solution, analyte, and/or internal standard to evaporate from the bottomportion of the microwave extraction vial and enter the headspace as avapor by diffusion and/or advection. In the embodiment where the samplecomprises an analyte but not an internal standard, preferably the vaporcomprises at least molecules of the analyte. In the embodiment where thesample comprises both an analyte and an internal standard, preferablythe vapor comprises at least molecules of the analyte and the internalstandard.

The vial also contains, in the headspace portion, a porous membrane bagthat encapsulates a liquid-phase extraction medium. In one embodiment,this porous membrane bag may be located above and out of contact fromthe solution and sample by a separation of 1-10 cm, preferably 5-10 cm,more preferably 6-9 cm.

In one embodiment, the porous membrane of the porous membrane bag maycomprise a polymer such as polypropylene, polyethylene, nylon,polyvinylidene fluoride, or polyethersulfone, preferably polypropyleneor polyethylene, even more preferably polypropylene. In an alternativeembodiment, more than one polymer may comprise the porous membrane. Forexample, the porous membrane may be composed of both polypropylene andpolyethylene with a polypropylene to polyethylene weight ratio range of1:10-10:1, preferably 1:5-5:1, more preferably 1:2-2:1,

In one embodiment, the porous membrane has a wall thickness of 10-200μm, preferably 15-100 μm, more preferably 25-50 μm. The porous membranemay have a pore diameter of 0.04-0.80 μm, preferably 0.1-0.5 μm, morepreferably 0.1-0.4 μm. The membrane may have a porosity of 40-90 vol %,preferably 50-85 vol %, more preferably 60-80 vol %. The liquid-phaseextraction medium may fill 70-100%, preferably 80-100%, more preferably90-100% of the pores exposed to the liquid-phase extraction medium fromthe interior of the membrane bag. Analyte contained in the headspacevapor may contact pores filled with liquid-phase extraction medium onthe exterior of the membrane bag, partition into the medium in the pore,and diffuse into the bag as a vapor extract. Preferably, compounds inthe sample or solution that are not of interest have low partitioninginto the vapor and/or into the liquid-phase extraction medium in thepores, and thus do not appreciably form a vapor extract in the membranebag. Preferably, the liquid-phase extraction medium and the vaporextract do not leak out of or evaporate from the membrane pores. Theliquid-phase extraction medium may be prevented from leaking out of thebag by its surface tension and/or interaction with the membranematerial. In an alternative embodiment, the liquid-phase extractionmedium does not fill the pores of the membrane, but vapor is still ableto diffuse through the pores and form a vapor extract within themembrane bag. In one embodiment, the membrane is pre-wetted with theliquid-phase extraction medium in order to fill the pores with themedium. In an alternative embodiment, the membrane is pre-wetted with aliquid phase that is a different compound than the liquid-phaseextraction medium contained in the membrane bag.

The porous membrane bag may comprise more than one piece of membrane,for instance, the bag may comprise both an inner and outer membranelayer. Where the bag comprises more than one membrane, the membranes maybe made of identical material. Alternatively, the membranes may be madeof different material, have different thicknesses, and/or have differentpore sizes.

The porous membrane bag may comprise a membrane in the shape of a tube,for example, a hollow fiber membrane, where the ends of the tube areclosed in order to contain the liquid-phase extraction medium. The edgesmay be closed by an adhesive, a clamp, a tie, or by heat sealing.Alternatively, the membrane may form a balloon shape around theliquid-phase extraction medium, with the membrane closed at one side, orwith the membrane edges tied at one point. Alternatively, the membranebag may form a rectangular pillow shape around the extraction medium. Inthis embodiment, the four edges may be sealed along each edge, or oneedge may be a fold in the membrane with the remaining edges being sealedalong each edge. In this pillow shape, the edges may measure 1-5 cm,preferably 2-4 cm, more preferably 2.2-3 cm in length, and the heightmay be 0.4-5 cm, preferably 0.8-4 cm, more preferably 0.8-2.8 cm.

In an alternative embodiment, the porous membrane bag is replaced by asingle porous membrane layer that holds the liquid-phase extractionmedium above the sample and solution. In another alternative embodiment,the porous membrane bag is replaced by a glass fit and/or supported by aglass fit that similarly holds the liquid-phase extraction medium abovethe sample and solution.

In another alternative embodiment, the liquid-phase extraction mediummay be contained in the headspace in a smaller vial that has an openingcovered by a porous membrane, which may allow for fragile membranematerials to be used in the microwave extraction assembly. This smallervial may be supported in the headspace by any of the previously ways ofsupporting the porous membrane bag, or the smaller vial may be attachedor built into an interior wall of the microwave extraction vial. Thesmaller vial may hold a volume of 0.05-5 mL, preferably 0.1-1 mL, morepreferably 0.25-0.75 mL of the liquid-phase extraction medium. Thesmaller vial may have a cylindrical or conical shape, and may bepositioned with the porous membrane on the top, side, or bottom. In thecase where the liquid-phase extraction medium may leak through themembrane, the smaller vial may be positioned with the porous membrane ontop. In one embodiment, the smaller vial is a tube with a porousmembrane secured across both openings.

In one embodiment the liquid-phase extraction medium is an organicsolvent, such as an alcohol, a polar aprotic solvent, a non-polarsolvent, or mixtures thereof. Preferably the boiling point of thesolvent or solvent mixture is greater than 50° C., preferably greaterthan 60° C., more preferably greater than 70° C., even more preferablygreater than 80° C. Examples of alcohols that can be used as theliquid-phase extraction medium include ethanol, methanol, 1-propanol,and 2-propanol; examples of polar aprotic solvents include acetone,dimethyl sulfoxide (DMSO), acetonitrile, dimethylformamide (DMF),tetrahydrofuran (THF), and ethyl acetate; examples of non-polar solventsinclude hexane, cyclohexane, heptane, decane, benzene, 1,4-dioxane,iso-octane, n-octane, nonane, and toluene. Preferably the organicsolvent is toluene, heptane, decane, iso-octane, or mixtures thereof.More preferably the organic solvent is toluene. The organic solvent mayfill at least 95%, preferably at least 98%, more preferably at least 99%of the total pore volume of the portion of membrane exposed to thesolvent. In an alternative embodiment, the extraction medium may be asolid-phase material, such as agarose, activated carbon, silica,alumina, magnesium silicate, or graphitized carbon black.

In one embodiment a single microwave extraction vial may comprise morethan one porous membrane bag, each enclosing a different solvent and/orcomprising a different type of membrane, for the purpose of extractingmore than one type of analyte from the sample. Preferably, thisembodiment may be used where two or more analytes in the sample havedifferent partitioning characteristics with a single liquid-phaseextraction medium. In this embodiment, different liquid-phase extractionmedia may be chosen to maximize the extraction efficiency of multipleanalyte types.

In one embodiment of the method, the weight of the sample relative tothe volume of the liquid-phase extraction medium is 0.04-100 g/mL,preferably 0.5-50 g/mL, more preferably 1-20 g/mL.

The porous membrane bag may hold a volume of 0.05-5 mL, preferably 0.1-1mL, more preferably 0.25-0.75 mL of the liquid-phase extraction medium.The membrane bag may enclose both the liquid-phase extraction medium anda gas. The gas may be present as a bubble or bubbles, and may compriseair, vaporized extraction medium, or any of the previously mentionedheadspace vapor components. The gas may result from filling and/orsealing the membrane bag with liquid-phase extraction medium or it maybe formed by heating the microwave extraction assembly. A gas in themembrane bag may comprise 0-50 vol %, preferably 0-10 vol %, morepreferably 0-1 vol % of the total enclosed volume of the membrane bag.

The porous membrane bag may be supported in the vial by a screen, amesh, or a ring placed inside the vial, or it may be supported by aprotrusion attached to or integral with an inner wall of the vial. Aring, such as a rubber O-ring or a non-metallic washer, may be anadvantageous support as it allows exposure of the membrane bag to vaporsabove and below in the headspace, and no modification to the microwaveextraction assembly is required beyond positioning the ring. Thus, themicrowave extraction assembly may comprise a standard laboratorymicrowave extraction vial without further alterations. Alternatively,the membrane bag may be supported from above, such as by a stringattached or tied to the membrane bag. This string may be attached to aninterior or exterior side of the microwave extraction vial, or it may beattached to the cap or closure of the microwave extraction vial. In arelated embodiment, the membrane of the membrane bag may be directlyattached to interior side of the closure or cap, such as by an adhesiveor a clamp. Preferably, the membrane bag is supported by a ring placedinside the vial, and in one embodiment, this ring comprises PTFE.

The method also involves extracting the analyte from the vapor toproduce a vapor extract within the liquid-phase extraction medium.

In one embodiment of the method, the extraction of the analyte occurs atthe same time as the microwave-heating and agitating, which means thatthe entire extraction method occurs in a single operation. For example,an analyte can be extracted from a sample and into the headspace vapor,and from the vapor into the liquid-phase extraction medium, withoutrequiring a researcher to perform those extraction steps separately orsubsequently. In addition, in the embodiment where the sample is derivedfrom an organism and the solution is an acid, the acid digestion of thesample is also contained within the extraction operation, meaning that aseparate sample preparation step such as cell membrane disruption is notrequired. Additionally, in this embodiment, parts of the analyte may besimultaneously partitioned to three locations within the microwaveextraction vial: in the sample and solution in the bottom portion, inthe vapor in the headspace portion, and within the liquid-phaseextraction medium. In another embodiment, the analyte may be limited todifferent locations in the microwave extraction assembly throughout themethod. For example, at the beginning of the heating and agitating step,the analyte may exist only in the bottom portion and in the vapor,without yet forming the vapor extract in the liquid-phase extractionmedium. Similarly, for example, prolonged microwave-heating maycompletely vaporize the sample and solution, leaving the analyte in onlythe vapor and the vapor extract. Likewise, an internal standard maysimultaneously exist in all three locations within the microwaveextraction vial, or may be limited to one or two locations.

The amount of analyte or internal standard in the phases of the sample,solution, vapor, and liquid-phase extraction medium may depend oninitial concentrations, the amount and rate of heating and agitating,and the partition coefficient of the analyte or internal standard amongthe different phases. Where two or more analytes are present, theirrelative concentrations may vary across different phases. Likewise,where one or more internal standards are present, the relativeconcentrations between an internal standard and an analyte, or betweentwo internal standards, may also vary across different phases.

The method also involves feeding the vapor extract to a gaschromatograph-mass spectrometer (GCMS) to detect and/or quantify theanalyte.

Following the vapor extraction, a vapor extract containing an analytemay be dissolved in the liquid-phase extraction medium. A syringe,cannula, or pipette may be used to puncture the membrane bag andwithdraw a portion or all of its contents. In an alternative embodiment,the membrane bag may be placed into a solvent that dissolves themembrane while retaining the analyte. The liquid-phase extraction mediummay be injected directly into a GCMS for analysis. Alternatively, theliquid-phase extraction medium may be diluted with an organic solvent orpurified before injection into the GCMS.

In one embodiment, a typical commercial GCMS may be used. The carriergas may be nitrogen, helium, and/or hydrogen. Preferably the carrier gasis helium with a purity of greater than 99.9 mol %, preferably greaterthan 99.99 mol %, more preferably greater than 99.999 mol %. Thestationary phase of the gas chromatography column may be comprised of amethyl siloxane (also known as methyl polysiloxane or dimethylpolysiloxane), phenyl polysiloxane, dimethyl arylene siloxane,cyanopropylmethyl polysiloxane, and/or trifluoropropylmethylpolysiloxane with a film thickness of 0.10-7 μm, preferably 0.15-1 μm,more preferably 0.2-0.5 μm. The column length may be 10-120 m,preferably 15-50 m, more preferably 25-40 m, with an inside diameter of0.08-0.60 mm, preferably 0.15-0.40 mm, more preferably 0.20-0.30 mm.

The parameters of a GCMS instrument and method of operation, includingbut not limited to flow rate, temperature, temperature gradient, runtime, pressure, sample injection, sample volume, ionization method,ionization energy, and scanning range may be adjusted by a person ofordinary skill in the art to account for differences in samples,equipment, and techniques.

The analyte may be detected by monitoring a known elution time and/orm/z (mass to charge ratio) for a positive signal as compared with ablank sample. For BDCM, 83, 85, and 129 m/z may be monitored in the massspectra; for TBM, 129, 173, and 252 m/z may be monitored; for TCM, 83and 85 m/z, may be monitored; for DBCM, 127, and 129 m/z may bemonitored, for DCP, 127 m/z may be monitored; and for TCP, 161 m/z maybe monitored. An internal standard's m/z may depend on its identity. Theanalyte may be quantified with a standard addition of an internalstandard. Examples of internal standards were discussed previously. Forquantitation, known concentrations of an internal standard may be addedto a sample that is divided into aliquots. These aliquots are eachextracted and measured by GCMS. Alternatively, aliquots of a vaporextract from a single trial could receive a standard addition. Thelinear response of the mass spectrometer counts per concentration ofinternal standard can be extrapolated to quantify an analyte.Additionally, more than one internal standard can be used in order tospan a range of molecular masses. Alternatively, standards may be usedto calibrate a GCMS prior to analyzing extracted samples.

In an alternative embodiment, gas chromatography may be used fordetection and/or quantitation of an analyte without using massspectrometry. In a related embodiment, the linear trend of the peakareas of the gas chromatogram may be used for quantitation. Generally, aperson of ordinary skill in the art may be able to determine theprocedure and calculations to quantify and/or detect an analyte based onGCMS data.

In one embodiment, the analyte is a trihalomethane or a haloketone. Inanother embodiment, the analyte is a trihalomethane or a haloketone, andmay be detected and/or quantified in the range of 0.02-200 ng,preferably 0.5-100 ng, more preferably 1-70 ng per g sample.Trihalomethanes and haloketones may have different limits of detection(LOD, or signal at limit of detection, or S_(LOD)) and different limitsof quantification (LOQ, or signal at limit of quantification, orS_(LOQ)), as defined by the equations S_(LOD)=S_(RB)+3σ_(RB) andS_(LOQ)=S_(RB)10σ_(RB), where S_(RB) is the signal of the reagent blankand σ_(RB) is the standard deviation of the reagent blank. In oneembodiment, TCM may have an LOD of at most 0.110 ng per g sample,preferably at most 0.10 ng/g, more preferably at most 0.02 ng/g, and anLOQ of at most 0.351 ng/g, preferably at most 0.2 ng/g, more preferablyat most 0.02 ng/g; BDCM may have an LOD of at most 0.051 ng per gsample, preferably at most 0.04 ng/g, more preferably at most 0.02 ng/g,and an LOQ of at most 0.175 ng/g, preferably 0.10 ng/g, more preferablyat most 0.02 ng/g; DCP may have an LOD of at most 0.085 ng per g sample,preferably at most 0.05 ng/g, more preferably at most 0.02 ng/g, and anLOQ of at most 0.263 ng/g, preferably at most 0.10 ng/g, more preferablyat most 0.02 ng/g; DBCM may have an LOD of at most 0.069 ng per gsample, preferably at most 0.04 ng/g, more preferably at most 0.02 ng/g,and an LOQ of at most 0.213 ng/g, preferably at most 0.10 ng/g, morepreferably at most 0.02 ng/g; TBM may have an LOD of at most 0.059 ngper g sample, preferably at most 0.04 ng/g, more preferably at most 0.02ng/g, and an LOQ of at most 0.182 ng/g, preferably at most 0.10 ng/g,more preferably at most 0.02 ng/g; TCP may have an LOD of at least 0.093ng per g sample, preferably at most 0.05 ng/g, more preferably at most0.02 ng/g, and an LOQ of at most 0.281 ng/g, preferably at most 0.10ng/g, more preferably at most 0.02 ng/g.

The examples below are intended to further illustrate methods ofmicrowave-assisted extraction of an analyte using a microwave extractionassembly and are not intended to limit the scope of the claims.

EXAMPLE 1 Experimental Setup Materials and Chemicals

A standard mixture of disinfection byproducts (DBPs) was obtained fromSigma Aldrich (Bellefonte, Pa., USA). This mixture contained THMs andHKs at 2000 μg mL⁻¹, which was prepared in methanol. Table 1 shows thephysical properties of target compounds [Guidechem,“2-Propanone,1,1-dichloro-513-88-2 properties reference,”http://www.guidechem.com/reference/dic-4656.html#id_-1077596984; WorldHealth Organization, “Trihalomethanes in Drinking-water, Backgrounddocument for development of WHO Guidelines for Drinking-water Quality,”http://www.who.int/water_sanitation_health/dwq/chemicals/en/trihalomethanes.pdf—eachincorporated herein by reference in its entirety].

By appropriate dilution of the stock solution of DBPs in the samesolvent, the working standard solutions were prepared on a daily basis.Required solvents were obtained from Supelco (Bellefonte, Pa., USA).Double deionized water was obtained from a Milli-Q system (Millipore,Bedford, Mass., USA). HNO₃ was obtained from Merck (Darmstadt, Germany).All glassware was washed with concentrated nitric acid, and then rinsedwith deionized water and acetone, and then dried at 100° C. for 1 h inan oven. A porous polypropylene membrane (2.5 cm×2.5 cm with 0.03 mmwall thickness) was obtained from Membrana (Wuppertal, Germany).

TABLE 1 Physical properties of target compounds. Physical properties TCMTBM DBCM BDCM DCP TCP Molecular weight (g mol⁻¹) 119.38 252.73 208.28163.8 126.96 161.41 Solubility at 20-25° C. (g L⁻¹) 7.5 3.1 1.0 3.3 6.37.4 Boiling point ° C. 61.3 149.5 119 90 117.3 — Vapor pressure at20-25° C. 21.28 0.75 2.0 6.67 — — (kPa) Melting point ° C. −63.2 8.3 —−57.1 — — Density at 20° C. (g/cm³) 1.484 2.90 2.38 1.98 1.291 — logK_(ow)* 1.97 2.38 2.08 1.88 0.20 1.12 *K_(ow) is the octanol-waterpartition coefficient

GC-MS Analysis

Analyses were carried out using GC-MS (Shimadzu technologies, QP 2010ultra system). An HP-1 methyl siloxane column (Shimadzu Rxi-5Sil MS;30.0 m×0.25 mm×0.25 μm thickness) was used. The carrier gas was heliumwith high purity (>99.999%), and a constant flow of 1.0 mL min⁻¹ wasused for analyzing samples. The following temperature program was usedfor the analyses: initial temperature of the column was 40° C., and itwas held for 5 min, and then increased to 150° C. at 10° C. mind andheld for 5 min. The total run time was 21 min. The injection port, ionsource, and interface temperatures were 200° C., 220° C., and 200° C.,respectively. For qualitative determinations, the scan mode was operatedfrom 50 to 550 m/z and for quantitative analysis selective ionmonitoring mode was used.

Fish & Green Alga Samples

Fresh fish samples (Striped Red Mullet) were obtained from fish marketsin Dammam, Eastern Province, Saudi Arabia. Fish samples were directlytransferred to an icebox (−4° C.) for temporary storage before reachingthe laboratory where they were stored at −18° C. prior to analysis.

Green alga samples were collected from the nearest desalination plant inJabil, Eastern Province, Saudi Arabia, and then used in the experimentsafter being air dried.

MA-HS-LPME Procedure

MA-HS-LPME experiments were carried out using a laboratory microwaveextraction system (Anton Paar, Graz, Austria, software version v1.52)with 16 closed polytetrafluoroethylene (PTFE) vessels. The vessel'svolume is 100 mL, and it can maintain a high temperature and pressure(240° C. and 40 bars).

Aqueous solutions are highly suitable for microwave digestion ofbiological samples, and tissues are easily digested by acidic or basicsolutions [Chan, C.-H., et al., Microwave-Assisted Extractions of ActiveIngredients from Plants. J. Chromatogr. A 2011, 1218,6213-6225—incorporated herein by reference in its entirety]. 3 grams ofbiological samples were transferred to clean microwave vessels equippedwith magnetic stir bars. 15 mL of 100 mM HNO₃ solution was added to eachvessel. The membrane bag was filled with 500 μl of an organic solventand heat sealed. This bag was suspended to certain depth in themicrowave vessel via a PTFE ring. FIG. 2 shows a schematic for the setupused.

EXAMPLE 2 Results and Discussion

MA-HS-LPME utilizes microwave energy for the fast heating of thesolutions by the rotation of the molecules through the migration of ionsand dipoles [Chan, et al., Microwave-Assisted Extractions of ActiveIngredients from Plants. J. Chromatogr. A 2011, 1218,6213-6225—incorporated herein by reference in its entirety]. Theefficiency of the extraction method is controlled by differentexperimental parameters, such as type of extraction solvent, depth ofthe membrane envelope inside microwave vessel, extraction temperature,sample weight, and extraction time.

Selection of Extraction Parameters

The selection of solvent plays a major role in MA-HS-LPME. Thedielectric constant and polarity of the solvent are important parametersto consider [Teo, C. C., et al., Development and Application ofMicrowave-Assisted Extraction Technique in Biological Sample Preparationfor Small Molecule Analysis. Metabolomics 2013, 9,1109-1128—incorporated herein by reference in its entirety]. Toluene,isooctane, heptane, and decane were selected as extraction solvents.FIG. 3 shows that toluene gave higher recoveries compared to othersolvents. That is due to two reasons: the polarity of toluene and itshigher dielectric constant, which when compared to other solvents is infollowing order: heptane<isooctane<decane<toluene [LSU MacromolecularStudies Group, “Dielectric Constant,”http://macro.Isu.edu/HowTo/solvents/Dielectric%20Constant%20.htm—incorporatedherein by reference in its entirety]. Thus, toluene was selected as anideal extraction solvent.

Depth of the solvent-containing porous membrane was investigated in therange of 1-7 cm above the samples. FIG. 4 reflects the effect ofmembrane depth on the extraction efficiency of THMs and HKs. As it canbe seen from FIG. 4, better extraction efficiency for all compounds wasachieved at a depth of 7 cm. Interaction of the vapors with the liquidphase and disturbance created by stronger stirring at lower depths canreduce the extraction efficiency of MA-HS-LPME. But as the vesselrepresents a closed system, homogenous vapor formation occurs at alldepths, so no significant differences were seen in the recovery ofanalytes.

The effect of the temperature on the extraction efficiency was evaluatedbetween 40 and 100° C. FIG. 5 illustrates the recoveries of targetanalytes at different extraction temperatures. Recoveries were increasedup to 80° C., and then leveled off. Temperatures below 80° C. were notenough to complete digestion of samples. By elevating the temperature,the extraction efficiency was improved. This increased extractionefficiency can be attributed to complete digestion of samples. However,at greater than 80° C., loss of solvent was observed. Thus, 80° C. wasselected as the ideal temperature and used in further studies.

The effect of simultaneous extraction and digestion time was examinedbetween 5 and 15 min. As it can be seen (FIG. 6), recoveries increasedup to 12 min and no further additional increments were observed afterthis time. This observation shows that 12 min is sufficient tocompletely digest the sample and vaporize the analytes.

Different sample sizes (1-5 grams) were investigated to determine theideal sample weight. FIG. 7 shows the influence of sample weight onrecoveries of analytes. As it can be clearly seen, the recoveryincreases upon increasing the sample weight. However, we did not attemptmore than 5 grams because it requires larger volumes of acid fordigestion, which increases sample digestion time.

Method Validation and Performance

In order to assess the matrix effect and to ensure more accuracy,matrix-matched calibration curves were used in this work by using thesample matrix spiked with analyte standards. The regression equationswere obtained by using 9 point standard concentrations (0.3, 0.5, 1, 5,10, 20, 40, 70, 100 ng/g) as the abscissa and the related chromatogrampeak areas as the vertical axis. It is presented in Table 2 that thelinearity in the concentration range of 0.3-100 ng/g was good forbrominated targets with 0.5-100 ng/g for the chlorinated ones with adetermination coefficient (R²) ranging from 0.9836 to 0.9954. Thesensitivity of the proposed method was determined by measuring thelimits of quantification (LOQ) and the limits of detection (LOD) oftested DBPs of the biota samples. Calculations were done according tothe guidelines of the American Chemical Society's Committee onEnvironmental Analytical Chemistry and IUPAC, whereS_(LOQ)=S_(RB)+10σ_(RB) and S_(LOD)=S_(RB)3σ_(RB), and where S_(LOQ),S_(LOD), and S_(RB) are the signal at the limit of quantification, atthe limit of detection, and of the reagent blank, respectively, whileσ_(RB) is the reagent blank's standard deviation. The method LOQ was inthe range from 0.175 to 0.351 ng/g while LODs of target analytes rangedfrom 0.051 to 0.110 ng/g. The relative standard deviations (RSDs) rangedbetween 1.1 and 6.8%. These results for the proposed method confirm thatMA-HS-LPME is suitable for the analysis of THMs and HKs at trace levelsin biological samples.

TABLE 2 Features of the MA-HS-LPME method. linear range LODs LOQs % RSDsNo. DBPs linear equation (ng/g) R² (ng/g) (ng/g) (n = 3) 1 TCM y =130.9.x + 1476.1 0.5-100 0.9875 0.110 0.351 1.12 2 BDCM y = 180.49x +3737.5 0.3-100 0.9887 0.051 0.175 3.64 3 DCP y = 59.129x + 404.190.5-100 0.9874 0.085 0.263 4.21 4 DBCM y = 140.5x + 6713.6 0.3-1000.9836 0.069 0.213 2.77 5 TBM y = 1883.6x + 9939.4 0.3-100 0.9884 0.0590.182 6.78 6 TCP y = 151.69.x + 994.31 0.5-100 0.9954 0.093 0.281 5.43

Real Sample Analysis

The developed MA-HS-LPME was applied for the detection of THMs and HKsin different biological samples. The previously determined experimentalconditions were used to quantitate DBPs in fish and green alga samples.The concentrations of target compounds that were detected are shown inTable 3. The results indicate that contents of THMs and HKs in biotasamples collected from different regions of Saudi Arabia were below thanUSEPA allowed limits [EPA Advice Note on Disinfection By-Products inDrinking Water, Advice Note No. 4. Version 2—incorporated herein byreference in its entirety]. Fish scales have accumulated these compoundsmore than tissues because of direct and constant contact with theexternal contaminated environment [King, R.P., et al., Aquaticenvironment perturbations and monitoring: African experience, USA, 2003166; Johal, M.S., et al., Sem study of the scales of freshwatersnakehead Charm punctatus (Bloch.) upon exposure to endosulfan. Bull.Environ. Contam. Toxicol. 1994, 52, 718721—each incorporated herein byreference in its entirety]. To assess the effect of matrix, real sampleswere spiked with 20 ng/g of target analytes. FIG. 8 depicts theextraction recoveries which range from 97.4 to 102.5%. FIG. 9A and FIG.9B present the GC-MS chromatograms of fish scale samples, unspiked andspiked, respectively, using the DSP labels from Table 2. Taken together,the chromatograms indicate no sample matrix interference using thismethod.

TABLE 3 Contents (ng/g) of the 6 analytes in the rested samples usingMA-HS-LPME. samples TCM BDCM DCP DBCM TBM TCP Fish scale 3.0 5.2 3.1 5.24.9 3.5 Fish tissue 1.9 3.9 2.0 3.5 3.9 2.1 Green alga 0.9 2.9 1.8 3.02.5 1.9

MA-HS-LPME, a novel and efficient extraction method, was originallydeveloped in this work. The feasibility of MA-HS-LPME was monitored byanalyzing the trace level quantitation of trihalomethanes andhaloketones in fish and green alga with GC-MS. Sample cleanup,extraction, and enrichment were done in one-step, which makes the samplepreparation much simpler and faster. Advantageous conditions were asfollowing: toluene as a solvent, membrane depth=7 cm, temperature=80°C., time=12 min, and sample weight=3 g. Low LODs, good linearity, andsatisfactory recoveries were obtained. MA-HS-LPME was an effectivetechnique in reducing the sample preparation time and solventconsumption. Microwave-assisted headspace liquid-phase microextractioncan be applied to the extraction of other classes of disinfectionbyproducts from complex sample matrices.

1: A method for extracting an analyte in a sample comprising:microwave-heating and agitating the sample and a solution in a microwaveextraction assembly comprising: a microwave extraction vial with abottom portion and a headspace portion and a porous membrane bag locatedin the headspace portion of the microwave extraction vial, said porousmembrane bag encapsulating a liquid-phase extraction medium, wherein thesample and solution are disposed in the bottom portion, the porousmembrane bag does not contact the sample and the solution, the porousmembrane bag and the headspace portion are also subjected to themicrowave-heating, and the microwave-heating and agitating produces avapor in the headspace portion; and extracting the analyte from thevapor to produce a vapor extract within the liquid-phase extractionmedium; and feeding the vapor extract to a gas chromatograph-massspectrometer (GCMS) to detect and/or quantify the analyte, wherein thevapor extract passes through a methyl siloxane gas chromatographiccolumn with a helium carrier gas.
 2. (canceled)
 3. (canceled) 4: Themethod of claim 1, wherein the weight of the sample relative to thevolume of the liquid-phase extraction medium is 0.04-100 g/mL. 5: Themethod of claim 1, wherein the, solution is an acid. 6: The method ofclaim 5, wherein the microwave-heating and agitating simultaneouslydigests the sample and extracts the analyte. 7: The method of claim 5,wherein the acid is at least one selected from the group consisting ofnitric acid, hydrochloric acid, hydrofluoric acid, sulfuric acid, andperchloric acid.
 8. (canceled) 9: The method of claim 1, wherein theporous membrane bag is positioned 1-10 cm above the sample. 10: Themethod of claim 1, wherein the weight of the sample relative to thevolume of the microwave extraction vial is 0.5-100 g/L. 11: The methodof claim 1, wherein the liquid-phase extraction medium is an organicsolvent. 12: The method of claim 11, wherein the organic solvent istoluene. 13: The method of claim 11, wherein the porous membrane bag ismade of polypropylene. 14: The method of claim 1, wherein the porousmembrane bag comprises a porous membrane having a 10-200 μm wallthickness. 15: The method of claim 1, wherein the microwave extractionassembly further comprises a ring within the headspace portion of themicrowave extraction vial to support the porous membrane bag. 16.(canceled) 17: The method of claim 1, wherein the sample and solutionare microwave-heated and stirred for 1-25 min. 18: The method of claim1, wherein the analyte is a trihalomethane or a haloketone. 19: Themethod of claim 2, wherein the analyte is a trihalomethane or ahaloketone, and the trihalomethane or haloketone is detected and/orquantified in the range of 0.02-200 ng trihalomethane or haloketone perg of sample. 20: The method of claim 18 wherein the trihalomethane isbromodichloromethane, tribromomethane, trichloromethane,dibromochloromethane, or combinations thereof, and the haloketone is1,1-dichloro-2-propanone, 1,1,1-trichloro-2-propanone, or both.